Necrosis has long been regarded as a passive, uncontrolled form of cell death. However, recent discoveries have challenged this notion providing evidence to show that necrosis is a tightly regulated cell death program with implication in various human pathological conditions [1, 2, 3, 4]. Similar to its cell death counterpart apoptosis, necrosis can be induced by an array of intrinsic and extrinsic stress signals [5]. The morphological manifestations that ensue have been well characterized which include increase in cell volume, swelling of the cellular organelles, rupturing of the cell membrane and subsequent spillage of their cellular contents into the surrounding environment [1]. Due to the lack of knowledge concerning the molecular mechanisms underlying necrotic cell death pathways, necrosis is often characterized by these features. However, insights into the necrotic process have recently emerged from studies conducted in the model organism Caenorhabditis elegans which have suggested that lysosomes are a major convergence point for necrotic stress pathways [17, 18]. In these studies, lysosome membrane permeabilization (LMP) and cysteine peptidase activity was triggered by a broad array of cellular insults. Interestingly, LMP and necrosis were blocked by overexpressing a single intracellular serine protease inhibitor (serpin), SRP-6, suggesting that necrosis is regulated by a proteases-driven mechanism. Our preliminary data indicates that similar necrotic pathways are employed in mammalian cells. In this proposal, we describe a research strategy aimed at identifying necrotic regulators of LMP and executioner proteases using two independent biochemical approaches. First, we will utilize a newly developed LMP activity assay to identify LMP regulators from human cell extracts using a series of purification steps followed by mass spectrometry. The second objective is to exploit the protease-inhibiting properties of human serpins in order to enrich for necrotic executioner proteases using an affinity tag pulldown method. A novel chemical compound, NB24, will be used to help stabilize serpin-protease complexes in the cell after necrosis induction. The protein composition of serpin-protein complexes will be analyzed via mass spectrometry. The function of all identified candidate proteins will be validated using cell-based survival assays to determine their function and mechanism of action during necrosis. Ultimately, the identification of these proteins will lead to the development of new therapeutic strategies to prevent LMP-induced necrosis, as well as provide the long-sought in vivo biomarkers of necrosis needed to further uncover necrotic pathways similar to how caspases have served for apoptosis research.